Aggregation-Induced Emission Probe for Study of the Bactericidal

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Aggregation-Induced Emission Probe for Study of the Bactericidal Mechanism of Antimicrobial Peptides Junjian Chen, Meng Gao, Lin Wang, Shiwu Li, Jingcai He, Anjun Qin, Li Ren, Yingjun Wang, and Ben Zhong Tang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18221 • Publication Date (Web): 22 Mar 2018 Downloaded from http://pubs.acs.org on March 22, 2018

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ACS Applied Materials & Interfaces

Aggregation-Induced Emission Probe for Study of the

Bactericidal

Mechanism

of

Antimicrobial

Peptides Junjian Chen,ab‡ Meng Gao,bc‡ Lin Wang,ab‡ Shiwu Li,bc Jingcai He,a Anjun Qin,bc Li Ren,b* Yingjun Wang,a* and Ben Zhong Tangcd* a

National Engineering Research Center for Tissue Restoration and Reconstruction, South China

University of Technology, Guangzhou 510006, China. E-mail: [email protected] b

School of Materials Science and Technology, South China University of Technology,

Guangzhou 510640, China. E-mail: [email protected] c

Guangdong Innovative Research Team, Center for Aggregation-Induced Emission, State Key

Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640, China d

Department of Chemistry and Hong Kong Branch of Chinese National Engineering Research

Center for Tissue Restoration and Reconstruction, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China. E-mail: [email protected] ‡ These authors contributed equally to this work.

ACS Paragon Plus Environment

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KEYWORDS: antimicrobial peptide, fluorescence imaging, aggregation-induced emission, real time monitoring, bactericidal mechanism

ABSTRACT. Multi-drug resistant bacterial infection has become one of the most serious threats to human health. Antimicrobial peptides (AMPs) have been identified as potential alternatives to antibiotics owing to their excellent bactericidal activity. However, the complicated bactericidal mechanism of AMPs is still poorly understood. Fluorescence imaging has many advantages in terms of dynamic monitoring, easy operation, and high sensitivity. In this study, we developed an aggregation-induced emission (AIE)-active probe AMP-2HBT by decorating of the antimicrobial peptide HHC36 (KRWWKWWRR) with an AIEgen of 2-(2-hydroxyphenyl)benzothiazole (HBT). This AIE-active probe exhibited an excellent light-up fluorescence after binding with bacteria, enabling a real-time monitoring of the binding process. Moreover, a similar timedependent bactericidal kinetics was observed for the AIE-active probe and HHC36 peptide, which indicated that the bactericidal activity of the peptide was not compromised by decorating with the AIEgen. The bactericidal mechanism of HHC36 peptide was further investigated by super-resolution fluorescence microscopy, transmission and scanning electron microscopy (TEM and SEM), which suggested that the probe tended to accumulate on the bacterial membrane and efficiently disrupt the membrane structure to kill both Gram-positive and -negative bacteria. This AIE-active probe thus provided a convenient tool to investigate the bactericidal mechanism of AMPs.

1. INTRODUCTION Bacterial infection has again become one of the most serious health threats, which is due to the fast growth of antibiotic resistance.1-6 Recently, the pursuit of new antimicrobial agents has


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become an urgent task. Among the emerging agents for bacterial killing, antimicrobial peptides (AMPs) are identified as potential alternatives to antibiotics,7-10 which can be due to their significant advantages in terms of broad-spectrum antimicrobial activity and low probability to generate resistance.11 To date, various AMPs from different natural hosts (insects, amphibians, and mammals, etc.) have been discovered and their bactericidal efficiency has been greatly improved through synthetic optimization.12 However, the bactericidal mechanisms of AMPs are still poorly understood.13-15 For example, HHC36 (KRWWKWWRR) peptide has shown an excellent bactericidal activity for both Gram-positive and -negative bacteria, but the exact mechanism is still unclear and several hypotheses have been proposed, such as membrane lysis, pore formation, disruption of surface proteins' function, and interaction with intracellular nucleic acids.16,17 To reveal the bactericidal mechanism of AMPs, it's highly desirable for real-time monitoring of the dynamic interaction process between AMPs and bacteria.18 Conventional imaging techniques, such as atomic force microscope (AFM), scanning electron microscope (SEM), and transmission electron microscope (TEM), all need complicated procedures to prepare samples and only provide steady-state information.19,20 As a result, they can't provide real-time information on the dynamic interactions between AMPs and bacteria. In contrary, fluorescence imaging can be used for dynamic and real-time observation with high sensitivity and easy operation.21,22 Fluorescence technique has thus been widely used to study the interactions between biomolecules, such as protein-protein interaction,23 peptide-receptor interaction,24 and aptamer-protein interaction.25 However, conventional fluorophores for study of the bactericidal mechanism of AMPs usually suffer from aggregation-caused quenching (ACQ) drawbacks.26-29 For example, the double-labelled AMPs with BODIPY fluorophore was reported to exhibit a signiﬁcantly weakened ﬂuorescence than mono-labelled AMPs, which is due to the


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self-quenching effect caused by the two neighbouring BODIPY molecules.27 Another example is the LAH4 peptide decorated with NBD fluorophore, which undergoes serious self-quenching after accumulation on the surface of lipid membranes.30 Because AMPs are prone to accumulate on the bacterial membrane at a high concentration to induce bactericidal effect,31-34 it is highly desirable to develop fluorescent probes without self-quenching drawbacks to study the bactericidal mechanism of AMPs. In contrary to ACQ, the aggregation-induced emission fluorogens (AIEgens) are almost nonemissive in dilute solution, but become highly emissive in the aggregate state.35 This phenomenon can be ascribed to the mechanism of restriction of intramolecular motion.36,37 Inspired by the significant advantages of AIEgens in bioimaging, such as free of self-quenching, high signal-to-noise ratio, and strong photostability,38-43 we herein developed an AIE-active probe AMP-2HBT through decorating the antimicrobial peptide HHC36 (KRWWKWWRR) with the AIEgen of 2-(2-hydroxyphenyl)benzothiazole (HBT) (Scheme 1). This AIE-active probe can be used for real-time monitoring of the binding process between antimicrobial peptide HHC36 and bacteria in a light-up and wash-free manner. Moreover, the membrane disruption mechanism of HHC36 peptide was revealed by super-resolution fluorescence microscopy (STORM), transmission and scanning electron microscopy (TEM and SEM).


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Scheme 1. Schematic illustration of AMP-2HBT for bacterial imaging and killing. 2. EXPERIMENTAL SECTION Materials and Chemicals. The antimicrobial peptide (HHC36, KRWWKWWRR) was purchased from China Peptides Co., Ltd. (Shanghai, China). 2,4-Dihydroxybenzaldehyde and ethyl 2-bromoacetate were purchased from Aladdin (Shanghai, China). 2-Aminobenzenethiol, Nhydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were purchased from Energy Chemical (Shanghai, China). DMSO, paraformaldehyde, trifluoroacetic acid, and acetonitrile were purchased from Sigma-Aldrich. Conc. HCl (37 wt. %), H2O2 (30 wt. %), ethanol, THF, and ethyl ether were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). Compound 3 was synthesized according to a method from the literature.44 All other chemicals were purchased and purified using standard procedures. Escherichia coli (E. coli, strain ATCC 15224) and Staphylococcus aureus (S. aureus, strain ATCC 6538) were purchased from VWR International, LLC (Padnor, USA). Nutrient broth and LB agar were purchased from Huankai Microbial Sci. & Tech. CO. LTD. (Guangdong, China).


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Characterization. The 1H and

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C NMR spectra were measured on a Bruker AV 500 NMR

spectrometer (Billerica, USA). The HPLC profiles were acquired on an Agilent 1260 Infinity LC (California, USA) with 0.1% TFA/acetonitrile and 0.1% TFA/H2O as the eluent. The highresolution mass spectra (HRMS) were measured on a Bruker maXis impact spectrometer (Billerica, USA). UV-Vis absorption spectra were recorded on a Shimadzu UV-2600 spectrophotometer (Kyoto, Japan). Photoluminescence (PL) spectra were taken on a Horiba Fluoromax-4 spectrofluorometer (Kyoto, Japan). Fluorescence quantum yields were measured by a Hamamatsu absolute PL quantum yield spectrometer C11347 Quantaurus_QY. The fluorescence assay for bacteria before and after treatment with AMP-2HBT was performed on Varioskan Flash (Thermo, USA) with 337 nm as the excitation wavelength. Flow cytometry was measured in a 96-well microtiter plate using Guava@ easyCyte HT (Millipore, USA) with 350 nm as the excitation wavelength. SEM images were acquired by a S-3700 scanning electron microscope (Hitachi, Japan). TEM images were acquired by a H-7650 transmission electron microscope (Hitachi, Japan). Synthesis of Ethyl 2-(4-(Benzo[d]thiazol-2-yl)-3-hydroxyphenoxy)acetate (4). Conc. HCl (37 wt.%, 100 mg) was added into an ethanol (15 mL) solution of ethyl 2-(4-formyl-3hydroxyphenoxy)acetate 3 (224 mg, 1.0 mmol) and 2-aminobenzenethiol (150 mg, 1.2 mmol), the mixture was stirred at room temperature for 10 min. H2O2 (30 wt.%, 113 mg, 1.0 mmol) was then added into the mixture, which was further stirred at room temperature for 2 h. After the reaction was completed, the solvent was removed under evaporation and the residue was recrystallized from ethanol to afford 4 as a yellow solid (230 mg, 70% yield). 1H NMR (CDCl3, 500 MHz): δ 12.68 (br s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 9.0 Hz, 1H), 7.49 (td, J1 = 7.5 Hz, J2 = 1.0 Hz, 1H), 7.38 (td, J1 = 7.5 Hz, J2 = 1.0 Hz, 1H), 6.59-6.56


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(m, 2H), 4.67 (s, 2H), 4.30 (q, J = 7.0 Hz, 2H), 1.32 (t, J = 7.0 Hz, 2H); 13C NMR (DMSO-d6, 125 MHz) δ 170.3, 166.0, 161.7, 158.5, 151.7, 134.0, 130.3, 126.9, 125.2, 122.4, 122.0, 112.4, 107.9, 102.4, 65.1, 14.5; HRMS (ESI): m/z [M + H]+ calcd for C17H16NO4S, 330.0800; found, 330.0795. Synthesis of 2-(4-(Benzo[d]thiazol-2-yl)-3-hydroxyphenoxy)acetic Acid (HBT-CO2H). Compound 4 (165 mg, 0.5 mmol) and NaOH (40 mg, 1.0 mmol) were added into a mixture of THF/H2O (5:2, v/v) solution, and the mixture was then heated under reflux for 3 h. After the reaction was completed, the mixture was cooled to room temperature and THF was removed under vacuum. Then, aq. HCl (1.0 M) was added in dropwise into the residue to yield a brown precipitation. After filtration, the obtained solid was sequentially washed with water, ethyl ether and further dried under vacuum to yield the pure product of compound 5 (78 mg, 52% yield). 1H NMR (DMSO-d6, 500 MHz): δ 13.09 (br s, 1H), 11.81 (br s, 1H), 11.81 (br s, 1H), 8.11 (d, J = 7.5 Hz, 1H), 8.04 (d, J = 9.0 Hz, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.52 (td, J1 = 7.5 Hz, J2 = 1.0 Hz, 1H), 7.42 (td, J1 = 7.5 Hz, J2 = 1.0 Hz, 1H), 6.62 (dd, J1 = 8.5 Hz, J2 = 2.5 Hz, 1H), 6.58 (d, J = 2.5 Hz, 1H), 4.76 (s, 2H); 13C NMR (DMSO-d6, 125 MHz) δ 170.3, 166.0, 161.7, 158.5, 151.7, 134.0, 130.3, 126.9, 125.2, 122.4, 122.0, 112.4, 107.9, 102.4, 65.1; HRMS (ESI): m/z [M + Na]+ calcd for C15H11NNaO4S, 324.0306; found, 324.0301. Synthesis of AMP-2HBT. NHS (6.9 mg, 60 µmol) and EDC (11.5 mg, 60 µmol) were added into the DMSO solution of compound 5 (6.0 mg, 20 µmol), the mixture was stirred at room temperature under nitrogen for 4 h. Then i-Pr2EtN (1.3 mg, 10 µmol) and HHC36 peptide (7.4 mg, 5 µmol) were added, the mixture was further stirred for 12 h. After completion of the reaction, the mixture was separated by HPLC (solvent A: water with 0.1% TFA, solvent B: CH3CN with 0.1% TFA) and lyophilized under vacuum to yield the desired product AMP-2HBT


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as a white solid in 36% yield (3.9 mg). HRMS (MALDI-TOF): m/z [M + H]+ calcd 2053.8872, found 2054.0993. Preparation of Stock Solution. The stock solutions of AMP-2HBT and HBT-CO2H were prepared by dissolving in DMSO solution (10 mM) and further stocked in the fridge at 4°C. LB nutrient broth and distilled water were sterilized by high temperature and stocked in the fridge at 4°C. Bacterial Culture. The single colony of E. coli was transferred into 6 mL of LB nutrient broth with shaking (150 rpm) at 37°C for 12 h, 0.1 mL of the bacterial solution was then inoculated in fresh LB nutrient broth (5 mL) and shaken (250 rpm) at 37°C for 6 h to achieve mid-log phase growth. The concentration of bacteria was measured by an optical density at 600 nm (OD600), and the scaler between the colony forming unit (CFU) of the bacteria and the OD600 was 1 OD600 = 1.65 × 109 CFU mL-1 The Antimicrobial Assay of AMP-2HBT. The MIC90 value of AMP-2HBT was determined by a broth protocol.45,46 The AMP-2HBT solution was added to the bacterial solution (5 × 105 CFU mL-1) to achieve a final concentration of 0 to 15 µM. After culturing for 18 h, the bacterial suspension was diluted 100, 101, 102, 103, and 104 times with PBS, and 10 µL of each solution were taken for spinning on agar plates. After culturing in the Mould Cultivation Cabinet for 15 h, the number of colonies on each agar plate was then counted. The bactericidal kinetics of AMP-2HBT and HHC36 peptide against S. aureus and E. coli was assessed using a time-dependent bactericidal assay. The AMP-2HBT was added to the bacteria solution (1.0 × 107 CFU mL-1) to achieve a final concentration of 10, 20, 50 and 100 µM.


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At 0, 15, 30, 45, 60, 120, 240 and 360 min, the bacterial solution was diluted 102, 103 and 104 times with PBS, and 10 µL of each solution were taken for spinning on agar plates to evaluate the viability of bacteria. Microbial Motility Measurement. We used digital holographic microscopy (DHM) to measure the inhibiting effect of the probe on the motility of E. coli, represented by the percentage of the actively-moving cells in the whole bacterial population within the observed region of interest.47 Briefly, for each of the multiple E. coli trajectories reconstructed by DHM, the mean squared displacement (MSD) is calculated as follows, MSD(∆t) = [|r(to + ∆t)-r(to)|2] Where r(t0) and r(t0 + t) are the 3D positions of an E. coli cell at times t0 and (t0 + t) respectively. The resultant MSD curve was fitted for each E. coli trajectory with at least 30 consecutive points: MSD(∆t) = D(∆t)v The power index υ was acquired for the first 10 % of the trajectory points. υ > 1 corresponded to active E. coli with super-diffusive motility. The motion of over 200 E. coli cells were recorded for each time. Fluorescence Imaging and Flow Cytometry Analysis. The bacteria were cultured in test tubes (BD Falcon, USA) with shaking (200 rpm) at 37°C for 5 h. Then, the bacteria were harvested by centrifuging at 3000 rpm for 3 min. After removal of the suspension, 1 mL of AMP-2HBT in distilled water at a concentration of 20 µM was added to the EP tube. After being dispersed, the bacteria were incubated at room temperature for desired time intervals, and then 3


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µL of the bacteria solution were added on the glass slide and covered by a coverslip. The fluorescence images were acquired by using an Eclipsc Ti-U (Nikon, Japan) with excitation wavelength at 340 nm. The super-resolution fluorescence images of bacteria were obtained on the Nikon STochastic Optical Reconstruction Microscopy (N-STORM, Nikon, Japan). The confocal lasing scanning microscopic (CLSM) images were obtained on the confocal microscope (Zeiss 710). Flow cytometry was carried out at desired time intervals (0, 15, 30, 45, and 60 min). λex = 350 nm, λem = 450−650 nm. 3. RESULTS AND DISCUSSION 3.1. Synthesis of AMP-2HBT. The synthetic route to AMP-2HBT is shown in Scheme 2. The reaction of 2,4-dihydroxybenzaldehyde 1 and ethyl 2-bromoacetate 2 first generated compound 3 in 68% yield. Compound 3 further reacted with 2-aminobenzenethiol in the presence of hydrogen chloride and hydrogen peroxide to generate HBT-CO2Et in 70% yield. HBT-CO2Et then underwent hydrolysis reaction with sodium hydroxide to afford HBT-CO2H in 52% yield. Structures of all the synthetic intermediates and HBT-CO2H were verified by NMR and HRMS analysis (Figure S1-S6). In the presence of EDC·HCl and NHS, HBT-CO2H was further reacted with antimicrobial peptide HHC36 (KRWWKWWRR) to afford the desired product AMP-2HBT in 36% yield, which was purified by HPLC and verified with HRMS (Figure S7-S8).


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SH

CHO

CHO

K2CO3

HO +

Br

CO 2Et

HO

NH 2

OH

O

1

N

2

N NaOH O

CO2 Et

Antimicrobial peptide (HHC36) EDC, NHS

OH

S

HBT

HBT NH

H2 N

NH NH

H N

NH

O

H N

N H

O

O

HN

O

H N

N H

O

N H

NH

HN

O

NH

NH

DMSO, RT

O COOH HBT-CO2 H, 52%

THF/H 2 O

4, 70%

H2 N

CO2Et

3, 68%

OH

S

HCl/H2O2 , EtOH, RT

MeCN, 80 oC

H N

O OH

N H

O

NH

O

NH

NH2

HN

NH2

AMP-2HBT HO N HBT

=

O

O

S

Scheme 2. Synthetic route to AMP-2HBT. 3.2. Photophysical Properties of AMP-2HBT. We then studied the photophysical properties of AMP-2HBT. As shown in Figure 1A, two main absorption peaks of AMP-2HBT in THF solution were observed at 287 and 337 nm, respectively, which could be ascribed to the benzothiazole moiety and the conjugation between benzothiazole and the substituted phenol moiety. We then measured the PL spectra of AMP-2HBT in THF solution and in the film state (Figure 1B). Almost no emission of AMP-2HBT was observed in the THF solution, while a strong emission was observed in the film state with a maximum emission peak at 489 nm and a large Stokes shift of 152 nm. The quantum yields of AMP-2HBT in the THF solution and in the film state were measured to be 0.9% and 20%, respectively, which clearly verified its AIE activity. The PL spectra of AMP-2HBT in the presence and absence of E. coli were also measured (Figure 1C). A significantly enhanced fluorescence was observed in the presence of bacteria, which suggested its promising applications for light-up and wash-free imaging.


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Figure 1. (A) Normalized UV spectrum of AMP-2HBT in THF solution; (B) PL spectra of AMP-2HBT in THF solution (black line) and in the solid state (red line). Inset: Photographs of AMP-2HBT in THF solution and in the film state taken under UV light (365 nm); (C) PL spectra of E. coli only, AMP-2HBT, and E. coli stained with AMP-2HBT in H2O/DMSO mixture (8:2, v/v). Excitation wavelength: 337 nm; Concentration of bacteria: 107 CFU mL-1; [AMP-2HBT] = 10 µM . 3.3. Antimicrobial Activity. The antimicrobial activity of AMP-2HBT was then investigated as a comparison to HHC36 peptide. With E. coli as an example, at concentrations of 10, 20, 50 and 100 µM, AMP-2HBT could kill 87.56%, 94.33%, 99.53% and 99.95% of E. coli, while HHC36 peptide could kill 84.25%, 94.64%, 99.68% and 99.84% of E. coli (Figure 2A). The MIC90 value of AMP-2HBT against E. coli was measured to be about 10 µM (Figure S9). The time-dependent killing kinetics of AMP-2HBT against Gram-positive S. aureus and Gramnegative E. coli was also measured (Figure S10). For S. aureus, the bacterial number decreased by approximately 3 orders of magnitude within 60 min at 20 µM of the probe, while all bacterial cells were killed within 60 min at 100 µM of the probe. For E. coli, the bacterial number decreased by approximately 3 orders of magnitude within 240 min at 20 µM of the probe, while all bacterial cells were killed within 360 min at 100 µM of the probe. Importantly, the


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bactericidal efficiency and time-dependent killing kinetics of AMP-2HBT were similar with HHC36 peptide, which indicated that the antimicrobial activity of HHC36 peptide was not compromised by decorating with AIEgens. We also conducted the bacterial inhibiting experiment of AMP-2HBT against E. coli (Figure 2B). Before addition of AMP-2HBT, 18%-32% of E. coli showed active movement, while its motility was almost completely inhibited within 2 min after addition of 20 µM probe. This experiment suggested that a low concentration of the probe could quickly inhibit the bacterial movement. 30 HHC36 peptide AMP-2HBT

A

100

B 25

75

Add AMP-2HBT

20

Activity (%)

Bacterial viability rate (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50

15

10 25 5

0

0 0

10

20

50

100

0

2

4

Concentration (µM)

6

8

10

12

14

16

18

Time (min)

Figure 2. (A) The antimicrobial ability of HHC36 peptide and AMP-2HBT against E. coli at different concentrations (0, 10, 20, 50 and 100 µM); (B) The time-dependent motility of E. coli in the presence of AMP-2HBT (20 µM); Concentration of bacteria: 107 CFU mL-1. 3.4. Real Time Monitoring of the Dynamic Binding Process. We then investigated the dynamic binding process of between AMP-2HBT and E. coli by fluorescence imaging (Figure 3). Before addition of AMP-2HBT, no fluorescence signal was observed for E. coli (Figure S11). After addition of AMP-2HBT, the fluorescent intensity of E. coli gradually increased with incubation time owing to the gradual accumulation of the probe on bacterial membrane (Figure


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3A-D). The flow cytometry experiment was also conducted and the bacterial staining ratio were found to be 14.0%, 40.9%, 65.0% and 85.0% at 15, 30, 45 and 60 min, respectively (Figure 3E). As a comparison, no fluorescence signal was observed for E. coli after incubation with the AIEgen of HBT-CO2H for 60 min (Figure S12), which unambiguously verified the binding ability of AMP-2HBT was based on the HHC36 peptide.

Figure 3. The fluorescence images of E. coli treated with AMP-2HBT at different times. (A) 15 min; (B) 30 min; (C) 45 min; (D) 60 min; (E) Flow cytometry histograms of E. coli treated with AMP-2HBT; Concentration of bacteria: 107 CFU mL-1; [AMP-2HBT] = 20 µM. 3.5. Super-resolution Fluorescence, TEM, and SEM Analysis. Because the fluorescence images obtained by confocal laser scanning microscope (CLSM) suffers from diffraction limit of light and can't precisely localize the spatial distribution of AMP-2HBT on bacterial membrane


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(Figure S13), we then conducted super-resolution fluorescence imaging with stochastic optical reconstruction microscopy (STORM).48 As shown in Figure 4A-B, a very strong but discontinuous fluorescence signal was observed on the bacterial membrane, which indicated the probe tended to aggregate on the bacterial membrane with a high density.49 We further studied the bacterial morphology changes by TEM and SEM. Before treatment with the probe, the TEM image showed that the bacteria are fulfilled with electron-dense materials and are enveloped with an intact cell membrane (Figure 4C). After treatment with AMP-2HBT, a hollow structure was observed, which suggested the intracellular substances leaked out from the disrupted membrane. Moreover, many bubbles were observed to protrude from the disrupted membrane. The SEM images showed that the E. coli membrane became very rough after treatment with AMP-2HBT (Figure 4E-F), suggesting that the bacterial membrane was efficiently destroyed.50,51 These imaging results suggested that the HHC36 peptide tended to aggregate on the bacterial membrane and efficiently disrupt the membrane structure to cause the flowing out of inner nucleic acids or proteins.


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Figure 4. (A-B) The super-resolution fluorescence images of E. coli after treatment with AMP2HBT. The TEM images of E. coli before (C) and after (D) treatment with AMP-2HBT. The bubbles protruded from the cell membrane were indicated by red arrows. The SEM images of E. coli before (E) and after (F) treatment with AMP-2HBT. [AMP-2HBT] = 20 µM. 4. CONCLUSION In summary, we have developed an AIE-active probe of AMP-2HBT for real-time monitoring of the bactericidal process of antimicrobial peptides. The AIE-active probe is especially suitable for high density labelling of bacterial membrane without self-quenching drawback, which provides an excellent opportunity for dynamic monitoring of the interaction process between AMPs and bacteria. It’s noteworthy that the AIEgen decorated on HHC36 peptide doesn't compromise the bactericidal activity. Based on super-resolution fluorescence imaging, TEM and SEM analysis, the excellent bactericidal activity of HHC36 peptide could be ascribed to its aggregation on the


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bacterial membrane and disruption of the membrane structure to cause the flowing out of inner nucleic acids or proteins. It's expected that this AIE-active probe would have broad applications for study of the bactericidal mechanism of antimicrobial peptides. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: NMR, HRMS, and HPLC spectra, the time-dependent killing kinetics, and fluorescence images of bacteria. AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] * E-mail: [email protected] * E-mail: [email protected] Author Contributions ‡These authors contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS


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This work was financially supported by the Science and Technology Planning Project of Guangzhou (Project No. 201607020015); National Science Foundation of China (Grant 51232002, 51620105009 and 21602063); Natural Science Foundation of Guangdong Province (2016A030313852 and 2016A030312002) and the Fundamental Research Funds for the Central Universities (2015ZY013 and 2015ZZ104). We also thank Prof. Xiangjun Gong and Prof. Bo Situ’s help for the microbial activity and super-resolution imaging experiments. REFERENCES (1) Hetrick, E. M.; Schoenfisch, M. H. Reducing Implant-Related Infections: Active Release Strategies. Chem. Soc. Rev. 2006, 35, 780-789. (2) Darouiche, R. O. Current Concepts - Treatment of Infections Associated with Surgical Implants. New Engl. J. Med. 2004, 350, 1422-1429. (3) del Pozo, J. L.; Patel, R. The Challenge of Treating Biofilm-Associated Bacterial Infection. Clin. Pharmacol. Ther. 2007, 82, 204-209. (4) Stewart, P. S. Mechanisms of Antibiotic Resistance in Bacterial Biofilms. Int. J. Med. Microbiol. 2002, 292, 107-113. (5) Fisher, J. F.; Meroueh, S. O.; Mobashery, S. Bacterial Resistance to Beta-Lactam Antibiotics: Compelling Opportunism, Compelling Opportunity. Chem. Rev. 2005, 105, 395-424. (6) Hoiby, N.; Bjarnsholt, T.; Givskov, M.; Molin, S.; Ciofu, O. Antibiotic Resistance of Bacterial Biofilms. Int. J. Antimicrob. Agents 2010, 35, 322-332.


18

Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(7) He, Y.; He, X. Molecular Design and Genetic Optimization of Antimicrobial Peptides Containing Unnatural Amino Acids against Antibiotic-Resistant Bacterial Infections. Biopolymers 2016, 106, 746-756. (8) Seo, M.-D.; Won, H.-S.; Kim, J.-H.; Mishig-Ochir, T.; Lee, B.-J. Antimicrobial Peptides for Therapeutic Applications: A Review. Molecules 2012, 17, 12276-12286. (9) Akram, A. R.; Avlonitis, N.; Lilienkampf, A.; Perez-Lopez, A. M.; McDonald, N.; Chankeshwara, S. V.; Scholefield, E.; Haslett, C.; Bradley, M.; Dhaliwal, K. A LabelledUbiquicidin Antimicrobial Peptide for Immediate in Situ Optical Detection of Live Bacteria in Human Alveolar Lung Tissue. Chem. Sci. 2015, 6, 6971-6979. (10) Kazemzadeh-Narbat, M.; Lai, B. F. L.; Ding, C.; Kizhakkedathu, J. N.; Hancock, R. E. W.; Wang, R. Multilayered Coating on Titanium for Controlled Release of Antimicrobial Peptides for the Prevention of Implant-Associated Infections. Biomaterials 2013, 34, 5969-5977. (11) Stach, M.; Siriwardena, T. N.; Koehler, T.; van Delden, C.; Darbre, T.; Reymond, J.-L. Combining Topology and Sequence Design for the Discovery of Potent Antimicrobial Peptide Dendrimers against Multidrug-Resistant Pseudomonas Aeruginosa. Angew. Chem. Int. Ed. 2014, 53, 12827-12831. (12) Zasloff, M. Antimicrobial Peptides of Multicellular Organisms. Nature 2002, 415, 389-395. (13) Gordon, Y. J.; Romanowski, E. G.; McDermott, A. M. A Review of Antimicrobial Peptides and Their Therapeutic Potential as Anti-Infective Drugs. Curr. Eye Res. 2005, 30, 505-515.


19


Page 20 of 26

(14) Kosciuczuk, E. M.; Lisowski, P.; Jarczak, J.; Strzalkowska, N.; Jozwik, A.; Horbanczuk, J.; Krzyzewski, J.; Zwierzchowski, L.; Bagnicka, E. Cathelicidins: Family of Antimicrobial Peptides. A Review. Mol. Biol. Rep. 2012, 39, 10957-10970. (15) Meneguetti, B. T.; Machado, L. d. S.; Oshiro, K. G. N.; Nogueira, M. L.; Carvalho, C. M. E.; Franco, O. L. Antimicrobial Peptides from Fruits and Their Potential Use as Biotechnological Tools-A Review and Outlook. Front. Microbiol. 2017, 7, 2136. (16) Nichols, M.; Kuljanin, M.; Nategholeslam, M.; Hoang, T.; Vafaei, S.; Tomberli, B.; Gray, C. G.; DeBruin, L.; Jelokhani-Niaraki, M. Dynamic Turn Conformation of A Short TryptophanRich Cationic Antimicrobial Peptide and Its Interaction with Phospholipid Membranes. J. Phys. Chem. B 2013, 117, 14697-14708. (17) Nategholeslam, M.; Vafaei, S.; Nichols, M.; Kuljanin, M.; Jelokhani-Niaraki, M.; Tomberli, B.; Gray, C. G. Structure of the Antimicrobial Peptide HHC-36 and Its Interaction with Model Cell Membranes. Biophys. J. 2012, 102, 397A-398A. (18) Bunschoten, A.; Welling, M. M.; Tennaat, M. F.; Sathekge, M.; van Leeuwen, F. W. B. Development and Prospects of Dedicated Tracers for the Molecular Imaging of Bacterial Infections. Bioconjugate Chem. 2013, 24, 1971-1989. (19) Anderson, R. C.; Haverkamp, R. G.; Yu, P. L. Investigation of Morphological Changes to Staphylococcus Aureus Induced by Ovine-Derived Antimicrobial Peptides Using TEM and AFM. FEMS Microbiol. Lett. 2004, 240, 105-110. (20) Hartmann, M.; Berditsch, M.; Hawecker, J.; Ardakani, M. F.; Gerthsen, D.; Ulrich, A. S. Damage of the Bacterial Cell Envelope by Antimicrobial Peptides Gramicidin S and PGLa as


20

Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Revealed by Transmission and Scanning Electron Microscopy. Antimicrob. Agents Chemother. 2010, 54, 3132-3142. (21) Rajasekhar, K.; Narayanaswamy, N.; Murugan, N. A.; Kuang, G.; Agren, H.; Govindaraju, T. A High Affinity Red Fluorescence and Colorimetric Probe for Amyloid Beta Aggregates. Sci. Rep. 2016, 6, 23668. (22) Jameson, L. P.; Smith, N. W.; Dzyuba, S. V. Dye-Binding Assays for Evaluation of the Effects of Small Molecule Inhibitors on Amyloid (A Beta) Self-Assembly. ACS Chem. Neurosci. 2012, 3, 807-819. (23) Wang, H.; Liu, J.; Han, A.; Xiao, N.; Xue, Z.; Wang, G.; Long, J.; Kong, D.; Liu, B.; Yang, Z.; Ding, D. Self-Assembly-Induced Far-Red/Near-Infrared Fluorescence Light-Up for Detecting and Visualizing Specific Protein-Peptide Interactions. ACS Nano 2014, 8, 1475-1484. (24) Shi, H.; Kwok, R. T. K.; Liu, J.; Xing, B.; Tang, B. Z.; Liu, B. Real-Time Monitoring of Cell Apoptosis and Drug Screening Using Fluorescent Light-Up Probe with AggregationInduced Emission Characteristics. J. Am. Chem. Soc. 2012, 134, 17972-17981. (25) Kong, R.-M.; Zhang, X.; Ding, L.; Yang, D.; Qu, F. Label-Free Fluorescence Turn-On Aptasensor for Prostate-Specific Antigen Sensing Based on Aggregation-Induced EmissionSilica Nanospheres. Anal. Bioanal. Chem. 2017, 409, 5757-5765. (26) Liu, C.; Gu, Y. Noninvasive Optical Imaging of Staphylococcus Aureus Infection in Vivo Using An Antimicrobial Peptide Fragment Based A Near-Infrared Fluorescent Probes. J. Innov. Opt. Health Sci. 2013, 6, 1350026.


21


Page 22 of 26

(27) Mendive-Tapia, L.; Zhao, C.; Akram, A. R.; Preciado, S.; Albericio, F.; Lee, M.; Serrels, A.; Kielland, N.; Read, N. D.; Lavilla, R.; Vendrell, M. Spacer-Free BODIPY Fluorogens in Antimicrobial Peptides for Direct Imaging of Fungal Infection in Human Tissue. Nat. Commun. 2016, 7, 10940. (28) Benincasa, M.; Pacor, S.; Gennaro, R.; Scocchi, M. Rapid and Reliable Detection of Antimicrobial Peptide Penetration into Gram-Negative Bacteria Based on Fluorescence Quenching. Antimicrob. Agents Chemother. 2009, 53, 3501-3504. (29) Park, C. B.; Kim, H. S.; Kim, S. C. Mechanism of Action of the Antimicrobial Peptide Buforin II: Buforin II Kills Microorganisms by Penetrating the Cell Membrane and Inhibiting Cellular Functions. Biochem. Biophys. Res. Commun. 1998, 244, 253-257. (30) Aisenbrey, C.; Bechinger, B. Molecular Packing of Amphipathic Peptides on the Surface of Lipid Membranes. Langmuir 2014, 30, 10374-10383. (31) Chen, J.; Zhu, Y.; Song, Y.; Wang, L.; Zhan, J.; He, J.; Zheng, J.; Zhong, C.; Shi, X.; Liu, S.; Ren, L.; Wang, Y. Preparation of An Antimicrobial Surface by Direct Assembly of Antimicrobial Peptide with Its Surface Binding Activity. J. Mater. Chem. B 2017, 5, 2407-2415. (32) Roversi, D.; Luca, V.; Aureli, S.; Park, Y.; Mangoni, M. L.; Stella, L. How Many Antimicrobial Peptide Molecules Kill A Bacterium? The Case of PMAP-23. ACS Chem. Biol. 2014, 9, 2003-2007. (33) Melo, M. N.; Ferre, R.; Castanho, M. A. R. B. OPINION Antimicrobial Peptides: Linking Partition, Activity and High Membrane-Bound Concentrations. Nat. Rev. Microbiol. 2009, 7, 245-250.


22

Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(34) Li, N. N.; Li, J. Z.; Liu, P.; Pranantyo, D.; Luo, L.; Chen, J. C.; Kang, E.-T.; Hu, X. F.; Li, C. M.; Xu, L. Q. An Antimicrobial Peptide with An Aggregation-Induced Emission (AIE) Luminogen for Studying Bacterial Membrane Interactions and Antibacterial Actions. Chem. Commun. 2017, 53, 3315-3318. (35) Luo, J. D.; Xie, Z. L.; Lam, J. W. Y.; Cheng, L.; Chen, H. Y.; Qiu, C. F.; Kwok, H. S.; Zhan, X. W.; Liu, Y. Q.; Zhu, D. B.; Tang, B. Z. Aggregation-Induced Emission of 1-methyl1,2,3,4,5-pentaphenylsilole. Chem. Commun. 2001, 18, 1740-1741. (36) Hong, Y.; Lam, J. W. Y.; Tang, B. Z. ChemInform Abstract: Aggregation-Induced Emission: Phenomenon, Mechanism and Applications. Chem. Commun. 2009, 29, 4332-4353. (37) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Aggregation-Induced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115, 11718-11940. (38) Kwok, R. T. K.; Leung, C. W. T.; Lam, J. W. Y.; Tang, B. Z. Biosensing by Luminogens with Aggregation-Induced Emission Characteristics. Chem. Soc. Rev. 2015, 44, 4228-4238. (39) Gao, M.; Wang, L.; Chen, J.; Li, S.; Lu, G.; Wang, L.; Wang, Y.; Ren, L.; Qin, A.; Tang, B. Z. Aggregation-Induced Emission Active Probe for Light-Up Detection of Anionic Surfactants and Wash-Free Bacterial Imaging. Chem.- Eur. J. 2016, 22, 5107-5112. (40) Gao, M.; Hu, Q.; Feng, G.; Tomczak, N.; Liu, R.; Xing, B.; Tang, B. Z.; Liu, B. A Multifunctional Probe with Aggregation-Induced Emission Characteristics for Selective Fluorescence Imaging and Photodynamic Killing of Bacteria Over Mammalian Cells. Adv. Healthc. Mater. 2015, 4, 659-663.


23


Page 24 of 26

(41) Feng, G.; Yuan, Y.; Fang, H.; Zhang, R.; Xing, B.; Zhang, G.; Zhang, D.; Liu, B. A LightUp Probe with Aggregation-Induced Emission Characteristics (AIE) for Selective Imaging, Naked-Eye Detection and Photodynamic Killing of Gram-Positive Bacteria. Chem. Commun. 2015, 51, 12490-12493. (42) Zhang, L.; Jiao, L.; Zhong, J.; Guan, W.; Lu, C.; Lighting Up the Interactions Between Bacteria and Surfactants with Aggregation-Induced Emission Characteristics. Mater. Chem. Front. 2017, 1, 1829-1835. (43) Zhang, P.; Nie, X.; Gao, M.; Zeng, F.; Qin, A.; Wu, S.; Tang, B. Z.; A highly selective fluorescent nanoprobe based on AIE and ESIPT for imaging hydrogen sulfide in live cells and zebrafish. Mater. Chem. Front. 2017, 1, 838-845. (44) Henary, M. M.; Fahrni, C. J. Excited State Intramolecular Proton Transfer and Metal Ion Complexation of 2-(2'-Hydroxyphenyl)benzazoles in Aqueous Solution. J. Phys. Chem. A 2002, 106, 5210-5220. (45) Pridmore, A.; Burch, D.; Lees, P. Determination of Minimum Inhibitory and Minimum Bactericidal

Concentrations

of

Tiamulin

against

Field

Isolates

of

Actinobacillus

Pleuropneumoniae. Vet. Microbiol.2011, 151, 409-412. (46) Brown, M. F.; Gupta, R. R.; Kuhn, M.; Flanagan, M. E.; Mitton-Fry, M. New Analyses of MIC90 Data to Aid Antimicrobial Drug Discovery. MedChemComm 2011, 2, 735-742. (47) Qi, M.; Gong, X.; Wu, B.; Zhang, G. Landing Dynamics of Swimming Bacteria on A Polymeric Surface: Effect of Surface Properties. Langmuir 2017, 33, 3525-3533.


24

Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(48) Rust M. J.; Bates M.; Zhuang X. Stochastic Optical Reconstruction Microscopy (STORM) Provides Sub-Diffraction-Limit Image Resolution. Nat. Meth. 2006, 3, 793-795. (49) Chen, R.; Mark, A. E. The Effect of Membrane Curvature on the Conformation of Antimicrobial Peptides: Implications for Binding and the Mechanism of Action. Eur. Biophys. J. Biophy. 2011, 40, 545-553. (50) Li, X.; Li, P.; Saravanan, R.; Basu A.; Mishra, B.; Lim, S. H.; Su, X.; Tambyah, P. A.; Leong, S. S. J. Antimicrobial Functionalization of Silicone Surfaces with Engineered Short Peptides Having Broad Spectrum Antimicrobial and Salt-Resistant Properties. Acta Biomater., 2014, 10, 258-266. (51) He, J.; Chen, J.; Hu, G.; Wang, L.; Zheng, J.; Zhan, J.; Zhu, Y.; Zhong, C.; Shi, X.; Liu, S.; Wang, Y.; Ren, L. Immobilization of an Antimicrobial Peptide on Silicon Surface with Stable Activity by Click Chemistry. J. Mater. Chem. B 2018, 6, 68-74.


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Table of Contents Graphic


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Aggregation-Induced Emission Probe for Study of the Bactericidal

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